1. Field of the Invention
The present invention relates generally to in vitro models of endothelial cells. More particularly, the invention relates to an in vitro model that simulates the characteristics of microvascular endothelial cells of the brain that constitute the blood-brain barrier.
2. Description of Related Art
The vertebrate brain has a unique capillary system unlike that of any other organ of the body. This unique capillary system has morphological and biochemical characteristics that make up the "blood-brain barrier" (BBB). The BBB acts to separate the brain interstitial space from the blood. This barrier prevents molecules in the blood that are neither lipophilic or transported by specific carrier proteins from entering the brain (Betz, A. L. et al., Ann. Rev. Physiol., 48:241 (1986); Pardridge, W. M., Ann. Rev. Pharmacol. Toxicol., 28:25 (1988)).
The characteristics of the brain capillaries that make up the BBB include: (a) high-resistance tight junctions between endothelial cells of the brain that block transport of molecules between cells; and (b) limited amount of transport across cells, as compared to that occurring in peripheral capillaries.
The tight junctions of the BBB prevent passive diffusion of molecules and ions around the endothelial cells. Thus, most hydrophilic drugs and peptides that gain ready access to other tissues of the body are barred from entry into the brain, or their rates of entry are low. Thus, at the BBB, the only substances that can readily pass from the luminal core of the capillary to the abluminal tissue that surround the capillary are those molecules for which selective transport systems exist in the endothelial cells, as well as compounds that are lipid soluble. Such compounds, because of their inherent lipophilicity, are able to intercalcate into the plasma membrane of endothelial cells and move to the abluminal side. These unique properties of the BBB have provided a major hindrance to the development of therapeutic agents directed toward diseases of the central nervous system (CNS), e.g., Alzheimer's disease and Parkinson's disease.
There are two general situations in which the ability to test for CNS entry of therapeutic agents is important. First, the increasing prevalence of CNS disorders and the introduction of new molecular biological and biochemical techniques to treat such disorders will lead to the development of new drugs that will be centrally active. These drugs must be tested for their ability to reach the brain, i.e., penetrate the BBB. Second, many drugs used to treat peripheral disorders have undesirable CNS side effects. As replacements for these drugs are developed, they will have to be screened for CNS penetration as well. Of course, the objective in that case is to develop peripherally-acting drugs that do not enter the brain.
Screening batteries of compounds for passage into the brain by conventional techniques is impractical. Generally, compounds are introduced into the carotid artery, and their concentration in the brain is then determined. This means that for each individual compound many animals must be injected and processed. While animal testing in vivo is important, it is not the optimal screening system when many compounds have to be examined.
Thus, it would be highly desireable to have an in vitro model of the BBB so as to be able efficiently and inexpensively to screen numerous drugs in a relatively short amount of time. The test system should closely simulate the morphological and physiological characteristics of the in vivo BBB in having tight junctions between cells and similar permeability characteristics, and should be composed of defined cell types.
Another desirable characteristic of an in vitro model is that it should provide a system for testing manipulations of the endothelial cells of a nature as to increase or decrease the passage of drugs from the blood side to the brain side of these cells.
Previous attempts to construct an in vitro model of the BBB have not met the criteria outlined above. Intact brain microvessels (Kumagai, A. K., J. Biol. Chem., 262:15214 (1987)) are likely to contain not only endothelial cells and astrocytes, but mast cells as well. Further, the limited volume and access to the lumen of microvessels precludes their use for vectorial transport studies, and therefore makes them suboptimal as a workable model for the BBB.
Several laboratories claim to have created a BBB in vitro model using brain capillary endothelial cells in the presence of standard growth media (Audus, K. L., et al., Ann. N.Y. Acad. Sci., 507:9 (1987); Van Bree, J. B. B. H., et al., Pharm. Res., 5:369 (1988); Hart, M. N., et al., J. Neuropath. Exp. Neurol., 46:141 (1987)). Cloned bovine brain capillary endothelial cells, grown on a permeable support of glutaraldehyde-treated collagen gel, have been reported to exhibit high transendothelial cell resistance (Rutten, M. J. et al., Brain Res., 425:301 (1987)). However, these studies have demonstrated only one or a few of the inherent morphological, biochemical and functional characteristics of brain capillaries, and the data derived from such systems are often conflicting, in part because in most studies the systems employed incompletely characterized populations of primary cell cultures or cell lines, and in part because the brain capillary endothelial cells were not grown in the proper milieu.
It is known that brain astrocytes influence the properties of brain capillary endothelial cells. Janzer et al. (Janzer, R. C., Nature, 325:253 (1987)) disclosed that neonatal rat brain type 1 astrocytes, cultured on filters and transplanted into the eyes of syngeneic animals or chick embryo chorioallantoic membranes, became vascularized by the endogenous endothelial cells, and caused the endothelial cells to exclude the dye, Evans blue.
Exclusion of Evans blue dye or other cationic dyes that bind to albumin is one property of endothelial cells in the brain. These results might be used to predict that astrocytes can cause endothelial cells to exhibit a generally low rate of macromolecular transport. They do not necessarily indicate, however, that the endothelial cells have been induced to form the high resistance tight junctions which are also characteristic of those cells in vivo.
Other in vitro studies have examined the effects of brain astrocytes on ultra-structural properties of endothelial cells. Brain astrocytes enhanced the frequency, length and complexity of tight junctions formed between cultured, brain-derived endothelial cells (Tao-Cheng, J.-H. et al., J. Neurosci., 7:3293 (1987)). Also, fourth passage rat brain capillary endothelial cell cultures, grown in rat brain astrocyte-conditioned medium on endothelial cell matrix-coated substrate, exhibited tight junction biogenesis (Arthur, F. E. et al., Dev. Brain Research, 36:155-9 (1987)). Both studies relied solely upon ultrastructural examination of individual groups of treated cells, but neglected to look at resistance of tight junctions.
Thus, an important need still exists for an in vitro model of a BBB that meets all of the criteria necessary for a model to simulate the in vivo situation: 1) a monolayer of endothelial cells essentially all of which are connected by tight junctions; 2) a diffusion barrier for components that do not ordinarily cross the BBB; and 3) a high transendothelial cell electrical resistance barrier indicating the presence of tight junctions that prevent passive diffusion of ions.